I am currently growing the THP-1 cell line for a research study on inflammatory markers and the cells don't seem to be growing very well. I am now in my third week of growing them and I have not seen them at confluence yet thus they haven't been subcultured yet either. I use RPMI-1640 from ATCC supplemented with 10% FBS and pen-strep. I have been changing media about every 2 days but have recently started letting them sit 3 days to try to let them grow more without being interrupted. I have also started seeing a few cells differentiate into macrophage like morphology taking on a more skewed tubular look. I know that some of these observations are "normal" and "expected" of this cell line in the first weeks (growing slowly, differentiation due to quality of media, growing in aggregates, etc.) but I am looking for any suggestions on how I can try to improve the ways in which I am culturing this line.
(Fyi, I am using a THP-1 cell culture protocol authored by Cordula Hirsch and have linked it as well for reviewal. I have made a few changes (pen-strep) but otherwise the protocol has been followed very closely).
Hello Wesley Quist
THP-1 cells are sensitive to cell density. The recommended seeding density is between 3-7 x 10^5 cells/ml. Generally, 5 x10^5 cells /ml is used..
THP-1 cells grow better in conditioned media, therefore whenever you passage the cells in the coming days, leave 1-2ml of old media and add fresh media to the culture.
If you have just thawed the cells recently, then you need to increase the FBS content to 20% for the first two to three passages, then you can switch over to 10% FBS for successive passaging. Try to increase the L-glutamine concentration from 0.2 mM to 2mM.
Sometimes cells may clump. But if they are growing fine and have a normal morphology it should not be a problem. You need to remove dead cells if they are present as sometimes these dead cells could be the cause of clumping.
Hope this helps!
Good Luck.
I have carried THP-1 cells in the past. According to the ATCC instructions, the culture medium is RPMI 1640 +10% FBS + 50 uM 2-mercaptoethanol. This is what I used.
Here's a link to the ATCC product information page: https://www.atcc.org/products/tib-202#detailed-product-information Look under the handling information tab.
Hi Wesley Quist,
When you first revive the THP-1 cell, are you using an untreated T-75 flask or an untreated T-25 flask? If you use T-75 flask, that explains why it grows slowly, cause it requires cell-to-cell interaction in order to grow and multiply well. I will recommend you to revive them in T-25 flask and let them incubate in a standing position instead of laying flat, as it increases the cell-to-cell interaction. Additional FBS helps to improve the cell viability and may help to grow better. Hope it helps you.
Seeding density is indeed very critical. However, I would start more at the lower end of 2 x10^5 cells/ml. Never let the grow to more than 1x10^6 /ml as this will trigger differentiation and stop them from proliferating. Adding 50 µM 2-mercaptoethanol as recommended by Gary Lee Gilmore is indeed very important as well to support the growth. If THP-1 are not cryopreserved perfectly they need some time (up to two weeks) to recover. What is your viability after thawing and 48 hours after seeding. You should except the viability is first decreasing if the cells have not been frozen in assay-ready quality.
If you are working with cells a lot you should check-out Cellseeker (https://www.cellseeker.org/). This is an easy and free to use software to inventory your cell collection.
Reading through that protocol you provided, I think you are probably seeding the cells at a very low concentration. I wouldn't go any lower than 2x105/ml. It is also very important to not let them get to a concentration higher than 106/ml. I remember when I was working with them, it took a long time to get enough cells for my experiments.
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